Project Summary The mosquito gut is a complex ecosystem containing a variety of bacterial taxa. These microbes play a crucial role in the biology of the insect. However, it remains unclear how microbes are acquired by mosquitoes and what life stages are important in this process. High throughput sequencing studies have shown the microbiome of the adult is highly variable but comprised of relatively few taxa. The microbiome of aquatic life stages is similar to the larval habitat, suggesting environmental factors influence acquisition. In addition, some bacteria can transition between mosquito life stages, suggesting they are highly adapted to the mosquito. Furthermore, it has been postulated that newly emerged adults imbibe larval water soon after emergence, which likely seed the gut microbiome, particularly with transient microbes. Together, these processes likely account for variation seen in the adult gut and suggest acquisition is a stochastic process. Not surprisingly, lab-reared mosquitoes have a vastly different microbiome compared to their field caught relatives. Given that the microbiome influences many mosquito phenotypes, including the ability of mosquitoes to transmit pathogens, spurious results may be obtained when studying processes with lab-reared individuals. Here, we will address these issues related to microbiome acquisition and differences between field and lab mosquitoes by exploiting a recently developed gnotobiotic system for rearing mosquitoes that allows for larvae to be reared with a defined environmental microbiome. In the first aim, we will examine how the larval habitat affects the adult gut microbiome. We will inoculate the larval habitat with a concentration gradient of a single bacterial species and observe how density in the water affects the density in the adult gut. Using two different fluorescently labeled bacteria (GFP vs mCherry), we will compare gut acquisition of a neutrally competing bacterium at similar or different larval water densities. For the last experiment of this aim, we will seed mosquito gut symbionts at different times in the developmental process to determine if the timing of inoculation influences the gut microbiome. In the second specific aim, we will exploit the gnotobiotic system to complete a microbiome transplantation between mosquito cohorts. We will optimize this procedure by transferring a simple microbiome that is comprised of three gut symbionts (Cedacea, Escherichia-Shigella and Pseudomonas) isolated from mosquitoes. Each of these bacteria will be tagged with a separate fluorescent protein that will allow culture- based validation. We will also develop a cryopreservation technique so the donor microbiome can be stored before reinfection. After optimizing the protocol with this tractable system, we will then undertake the transfer of a more complex microbiome using a donor microbiome sourced from Ae. aegypti collected from the field in Galveston and transferred this microbiome into a lab Ae. aegypti (Galveston strain). These studies will provide a more complete understanding of host acquisition of the microbiome and develop important tools for manipulating the microbiome, which will be significant for further studies examining host-microbe interactions.